Redox mediators

ABSTRACT

The present invention relates to ruthenium and osmium complexes of Formula [M(A) w (B) x C y ] m (X z ) n , per se and the use of ruthenium and osmium complexes of Formula I as redox mediators in electrochemical biosensors.

The present invention relates to novel ruthenium and osmium complexes,the use of ruthenium and osmium complexes as a redox mediator or in abiosensor. In particular, the present invention relates to the use of aruthenium complex having an overall charge on the ruthenium-containingspecies less than 3+in the ruthenium (III) state as a redox mediator.

A biosensor is an analytical tool combining a biochemical recognitioncomponent or sensing element with a physical transducer. A biosensor hasbroad application in fields as diverse as personal health monitoring,environmental screening and monitoring, bioprocess monitoring and withinthe food and beverage industry. Biosensors offer the convenience andfacility of distributed measurement ie the potential ability to take theassay to the point of concern or care. A properly designed andmanufactured biosensor may be conveniently mass-produced.

The biological sensing element may be an enzyme, antibody, DNA sequenceor microorganism which serves (for example) to catalyze selectively areaction or facilitate a binding event. The selectivity allows for theoperation of the biosensor in a complex sample matrix (eg a body fluid).The transducer converts the biochemical event into a measurable signalthereby providing the means for detecting it. The measurable signal maybe a spectral change caused by the production or consumption of theproduct or substrate of an enzymatic reaction or a mass changeassociated with biochemical complexation. The transducer may beoptically-based to measure optical absorption, fluorescence orrefractive index. The transducer may be mass-based to measure a changein mass that accompanies a binding reaction. The transducer may bethermally-based to measure a change in enthalpy (heat) or amperometry.The transducer may be impedance-based to measure a change in anelectrical property that accompanies the interaction of ananalyte/bio-recognition layer.

Enzyme-based biosensors are used widely in the detection of analytes inclinical, environmental, agricultural and biotechnological applications.They offer specificity, sensitivity and operate under mild conditions.Analytes that can be measured in clinical assays of fluids of the humanbody include (for example) glucose, lactate, cholesterol, bilirubin andamino acids. Levels of these analytes in biological fluids (such asblood) are important for the diagnosis and monitoring of diseases. Thereare however disadvantages associated with use of biosensors whichinclude the vulnerability of the transducer to foulants andinterferences.

Sensors which generally exploit enzyme-based systems are provided aseither point-of-care or over-the-counter devices. They can be used totest fresh, unmodified, whole blood finger prick samples in order todetermine the concentrations of total cholesterol, triglycerides, HDLand LDL within (for example) 1 to 5 minutes of adding the sample to adevice. These four parameters in combination have been clinically provento give a very good indication of the risk of heart disease in adults.It is well, known that high cholesterol is asymptomatic and it isrecommended that an adult should have a test to assess their risk. Iftheir risk is found to be high, it may be significantly reduced bycorrect management of diet alone or in combination with theadministration of a therapeutic drug.

An electrochemical assay is typically performed in a cell with two orthree electrodes which include at least one measuring or workingelectrode and one reference electrode. In a three electrode system, thethird electrode is a counter-electrode. In a two electrode system, thereference electrode also serves as the counter-electrode. The electrodesare connected through a circuit such as a potentiostat. The measuring orworking electrode is a carbon or metal conductor or semiconductor.

In an example of an enzyme-based biosensor, there is utilised anelectrochemical assay to detect an analyte. Use is made of a change inthe oxidation state of a mediator which interacts with an enzyme whichhas reacted with the analyte. The oxidation state of the mediator ischosen so that it interacts with the enzyme on addition of thesubstrate. The analyte reacts with a stoichiometric concentration of themediator via the enzyme. This causes the mediator to be oxidised orreduced (depending on the enzymatic reaction) and this change can bemeasured by determining the current generated at a given potential or bydetermining the potential at a given current.

In a further example of an enzyme-based biosensor, a sufficiently largevoltage passed to the working electrode causes a redox enzyme to beelectrooxidized or electroreduced. The enzyme is specific to the analyteto be detected or to a product of the analyte. The turnover rate of theenzyme is typically related (eg linearly) to the concentration of theanalyte itself or to its product in the test solution.

The electrooxidation or electroreduction of the enzyme is oftenfacilitated by the presence of a redox mediator in the solution or onthe electrode. The redox mediator generally assists in the electricalcommunication between the working electrode and the enzyme. The redoxmediator can be dissolved in the fluid to be analyzed which is inelectrolytic contact with the electrodes. A useful device may be made(for example) by coating an electrode with a film that includes a redoxmediator and an enzyme catalytically specific to the desired analyte orits products. A diffusional redox mediator which can be soluble orinsoluble in water functions by shuttling electrons between (forexample) the enzyme and the electrode. When the substrate of the enzymeis electrooxidized, the redox mediator transports electrons from thesubstrate-reduced enzyme to the electrode. When the substrate iselectroreduced, the redox mediator transports electrons from theelectrode to the substrate-oxidized enzyme.

Conventional enzyme-based electrochemical sensors have employed a numberof redox mediators including monomeric ferrocenes, quinoid-compounds(such as quinines eg benzoquinones), nickel cyclamates and rutheniumamines. For the most part, these redox mediators have one or more of thefollowing limitations:

-   -   the solubility of the redox mediator in the test solutions is        low,    -   the chemical, light, thermal or pH stability of the redox        mediator is poor,    -   the redox mediator does not exchange electrons rapidly enough        with the enzyme or the electrode or both.

Additionally the oxidation potential of many of these redox mediators isso high that at the potential where the reduced mediator iselectrooxidized on the electrode, solution components other than theanalyte are also electrooxidized. In other cases, the reductionpotential is so low that the solution components (such as for exampledissolved oxygen) are also rapidly electroreduced. As a result, thesensor utilizing the mediator is not sufficiently specific.

Ruthenium-based complexes have previously been utilised as redoxmediators in reactions containing (for example) cholesteroldehydrogenase. For example, a [Ru¹¹(NH₃)₆)²⁺ species is converted to[Ru^(III)(NH₃)₆]³⁺ at an electrode poised at a suitable potential. Thecurrent is proportional to the amount of [Ru^(II)(NH₃)₆]²⁺ speciesformed via the enzymatic reaction. However a highly-charged species suchas [Ru^(III)(NH₃)₆]³⁺ forms (to a greater or lesser extent) complexeswhich are usually in the form of ion-pairs with negatively-chargedgroups on enzymes and the electrode surface. This impedes the reactionsnecessary for the analytical process to occur effectively andefficiently.

It would therefore be desirable to utilise a redox mediator which formsless strong complexes or none at all with the components of theanalytical mixture and the electrode so that measured responses are morereliable, stable and reproducible.

According to a first aspect of the present invention there is providedthe use of a complex of Formula I

[M(A)_(w)(B)_(x)(C)_(y)](C)_(y)]^(m)(X^(z))_(n)   Formula I

(wherein

-   -   M is ruthenium or osmium and has an oxidation state of 0, 1, 2,        3 or 4;    -   each of w, x, and y is an integer independently selected from        the integers 1 to 4;    -   m is an integer selected from the integers −5 to +4;    -   n is an integer selected from selected from the integers 1 to 5    -   z is an integer selected from the integers −2 to +1;    -   A is a monodentate 5- or 6-membered aromatic ligand containing        1, 2 or 3 nitrogen atoms which is optionally substituted by 1 to        8 substituents each selected from the group consisting of        substituted or unsubstituted alkyl, alkenyl, or aryl groups, —F,        —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, aryl,        alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH,        alkoxy, —NH₂, alkylamino, dialkylamino, alkanoylamino,        arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino,        alkoxyamino and alkylthio or A is NCS;    -   B is a bi-, tri-, tetra-, penta- or hexadentate ligand which is        linear having the formula R¹RN(C₂H₄NR)_(w)R¹ or cyclic having        the formula (RNC₂H₄)_(v), (RNC₂H₄)_(p)(RNC₃H₆)_(q) or        [(RNC₂H₄)(RNC₃H₆)]_(s), wherein        -   w is an integer selected from the integers 1-5,        -   v is an integer selected from the integers 3-6,        -   each of p and q is an integer independently selected from            the integers 1-3 whereby the sum of p and q is 4, 5 or 6,        -   s is either 2 or 3 and        -   each of R and R^(I) is independently hydrogen or alkyl;    -   C is a ligand other than B; and    -   X is a counter ion,

wherein the number of coordinating atoms is 6) with the exception of[Ru^(III)(Me₃tacn)(acac)(py))(NO₃)₂) as a redox mediator.

In a preferred embodiment of the use according to the invention, in thecomplex of Formula I:

-   -   A is a monodentate 5- or 6-membered aromatic ligand containing        1, 2 or 3 nitrogen atoms which is optionally substituted by 1 to        8 substituents each selected from the group consisting of        substituted or unsubstituted alkyl, alkenyl, or aryl groups, —F,        —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, aryl,        alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH,        alkoxy, —NH₂, alkylamino, dialkylamino, alkanoylamino,        arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino,        alkoxyamino and alkylthio; and    -   C is a ligand other than A or B.

The ligand A in the complex of Formula I may be selected from the groupconsisting of NCS, imidazole, pyrazole, thiazole, oxazole, isoquinoline,substituted pyridyl (eg 3- and/or 4-substituted pyridyl) and isomersthereof.

The ligand A in the complex of Formula I may be selected from the groupconsisting of imidazole, pyrazole, thiazole, oxazole and isomersthereof.

The ligand A in the complex of Formula I may be or contain a 5- or6-membered aromatic ligand containing 3 nitrogen heteroatoms. The ligandA in the complex of Formula I is preferably triazine or triazole.

The ligand A in the complex of Formula I may be guanine or adenine.

The ligand A in the complex of Formula I may be substituted by one ormore substituents selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl, halogen, carboxy, amino,C₁₋₆-alkylamino, C₁₋₆-dialkylamino and hydroxyl.

The ligand A in the complex of Formula I may be _substituted by one ormore substituents selected from the group consisting of C₁-C₆ alkyl,C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl and halogen. Preferably theligand A in the complex of Formula I is substituted by one or moresubstituents selected from the group consisting of methyl, ethyl,propyl, iso-propyl, butyl, t-butyl, methoxy, ethoxy, ethenyl, propenyl,butenyl, ethynyl and propynyl.

The ligand B in the complex of Formula I may be a bi-, tri- ortetra-dentate ligand which may be linear having the formulaR¹RN(C₂H₄NR)_(r)R^(I) or cyclic having the formula (RNC₂H₄)_(v),(RNC₂H₄)_(p)(RNC₃H₆)_(q) or [(RNC₂H₄)(RNC₃H₆)]_(s), wherein r is aninteger selected from the integers 1-3, v is 3 or 4, each of p and q isan integer independently selected from the integers 1-3 whereby the sumof p and q is 4 and s is 2 or 3.

Preferably the ligand B in the complex of Formula I is a tri- ortetra-dentate ligand which is cyclic having the formula (RNC₂H₄)_(v),wherein v is 3 or 4.

The ligand B in the complex of Formula I may be1,4,7-trimethyl-1,4,7-triazacyclononane,1,4,8,11-tetramethyl-1,4,8,11-tetra-azacyclotetradecane,1,1,4,7,10,10-hexamethyltriethylenetetramine,1,2-dimethylethylenediamine or 1,1,2,2-tetramethylethylenediamine.

The ligand B in the complex of Formula I may be1,4,7-trimethyl-1,4,7-triazacyclononane,1,1,4,7,10,10-hexamethyltriethylenetetramine,1,2-dimethylethylenediamine or 1,1,2,2-tetramethylethylenediamine.

A preferred ligand B in the complex of Formula I is1,4,7-trimethyl-1,4,7-triazacyclononane.

The ligand C in the complex of Formula I may be selected from the groupconsisting of an amine ligand (such as NH₃), CO, CN, NCS, a halogen,acetylacetonate (acac), 3-bromo-acetylacetonate (Bracac), oxalate,troplone, pyridine and 5-chloro-8-hydroxyquinoline.

The ligand C in the complex of Formula I may be selected from the groupconsisting of an amine ligand (such as NH₃), CO, CN, a halogen,acetylacetonate (acac), 3-bromo-acetylacetonate (Bracac), oxalate,pyridine and 5-chloro-8-hydroxyquinoline.

A preferred ligand C in the complex of Formula I is acac.

The ligands A, B and C in the complex of Formula I may be bidentate. Thegeometry of the complex of Formula I may be cis or trans.

The oxidation state of the metal in the complex of Formula I may be 2+,3+ or 4+. The oxidation state of the metal in the complex of Formula Iis preferably 3+.

The ligands A, B and C may be selected such that the overall charge onthe complex of Formula I is selected from the group consisting of +3,+2, +1, 0, −1, −2 and −3.

The counterion X in the complex of Formula I may be F⁻, Cl⁻, Br⁻, I⁻,NO₃ ⁻, NH₄ ⁺, NR₄ ⁺, PF₆ ⁻, CF₃SO₃ ⁻, SO₄ ²⁻, ClO₄ ⁻, K⁺, Na⁺, Li⁺ or acombination thereof.

The complex of Formula I used as a redox mediator in accordance with thepresent invention may be[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-methylpyridine)]Cl₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3-chloropyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(isonicotinamide)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)pyrazine](NO3)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane(acac)(4-methoxypyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-dimethylaminopyridine)1(NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-t-butyl-pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(isoquinoline)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(tropolone)(pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(tropolone)(4-t-butyl-pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3,4-dimethylpyridine)](CF₃SO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3-hydroxypyridine)](NO₃)₂or[Ru^(III)(1,4,8,11-tetramethyl-1,4,8,11-tetra-azacyclotetradecane)(NCS)₂](ClO₄).

The complex of Formula I used as a redox mediator in accordance with thepresent invention may be[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](PF₆)₂,or[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](NO₃)₂.

The complex of Formula I may be used as a redox mediator in accordancewith the present invention in an electrochemical sensor. Theelectrochemical sensor may include a microband electrode. Theelectrochemical sensor may be an electrochemical biosensor. Theelectrochemical sensor may be used to detect an analyte in a bodilyfluid, environmental sample, food, beverage, veterinary sample orpharmaceutical.

The complex of Formula I may be used as a redox mediator in accordancewith the invention at a pH of 6 to 10, preferably at a pH of 7 to 9.

According to a second aspect of the present invention there is providedthe use of a ruthenium complex of Formula I as hereinbefore defined in abiosensor.

The biosensor may be used with a compatible biochemical analyte. Theanalyte may be found in a biological fluid. The analyte may be selectedfrom the group consisting of an enzyme, enzyme substrate, antigen,antibody, nucleic acid sequence, cholesterol, cholesterol ester,lipoprotein, triglyceride and microorganism.

The complex of Formula I as hereinbefore defined may be used in abiosensor which consists of (for example) a strip with four reagentwells and a common pseudo reference. Each well may have a tubularmicro-band working electrode. The sensing component of the strip may beprovided by drying different specially formulated reagents comprising atleast an enzyme and a mediator that is capable of interacting withspecific analytes in the test sample in each well. Since differentreagents may be added and dried to each well, it is possible to completemulti-analyte testing using a single test sample. The number of wells isvariable and so the number of unique tests is variable. For example,sensors having between 1 and 6 wells may be used.

The complex of Formula 1 as hereinbefore defined may be used in abiosensor which consists of (for example) a conventional microelectrodewhich typically has a working microelectrode and a reference electrode.The working electrode may be made of palladium, platinum, gold orcarbon. The counter electrode may be typically carbon, Ag/AgCl,Ag/Ag₂SO₄, palladium, gold, platinum, Cu/CuSO₄, Hg/HgO, Hg/HgCl₂,Hg/HgSO₄ or Zn/ZnSO₄. Preferably the working electrode is in a wall of areceptacle forming the microelectrode. Examples of microelectrodes whichcan be used in the present invention are those disclosed inWO-A-03/097860.

According to a third aspect of the present invention there is provided adetection system for measuring an analyte comprising:

-   -   (a) contacting a sample which contains the analyte with a        solution containing a redox mediator according to Formula I as        defined hereinbefore;    -   (b) incubating the contacted sample under conditions that cause        the enzyme to act on the analyte;    -   (c) subjecting the incubated sample of step (b) to conditions        which result in a change in a measurable signal; and    -   (d) measuring the measurable signal.

The measurable signal may be an electrochemical, colourimetric, thermal,impedometric, capacitive or spectroscopic signal. The measurable signalmay be an electrochemical signal measured at a microband electrode. Theelectrochemical signal may be detected using a microband electrode in anamperometric detection method.

According to a fourth aspect of the present invention there is provideda complex according to Formula I

[M(A)_(w)(B)_(x)(C)_(y)]^(m)(X^(z))_(n)   Formula I

(wherein

-   -   M is ruthenium or osmium and has an oxidation state of 0, 1, 2,        3 or 4;    -   each of w, x, and y is an integer independently selected from        the integers 1 to 4;    -   m is an integer selected from the integers −5 to +4;    -   n is an integer selected from selected from the integers 1 to 5    -   z is an integer selected from the integers −2 to +1;    -   A is a monodentate 5- or 6-membered aromatic ligand containing        1, 2 or 3 nitrogen atoms which is optionally substituted by 1 to        8 substituents each selected from the group consisting of        substituted or unsubstituted alkyl, alkenyl, or aryl groups, —F,        —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, aryl,        alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH,        alkoxy, —NH₂, alkylamino, dialkylamino, alkanoylamino,        arylcarboxamido, hydrazino, alkylhydrazino, hydroxylamino,        alkoxyamino and alkylthio;    -   B is a bi-, tri-, tetra-, penta- or hexadentate ligand which is        linear having the formula R¹RN(C₂H₄NR)_(w)R¹ or cyclic having        the formula (RNC₂H₄)_(v), (RNC₂H₄)_(p)(RNC₃H₆)_(q) or        [(RNC₂H₄)(RNC₃H₆)]_(s), wherein        -   w is an integer selected from the integers 1-5,        -   v is an integer selected from the integers 3-6,        -   each of p and q is an integer independently selected from            the integers 1-3 whereby the sum of p and q is 4, 5 or 6,        -   s is either 2 or 3 and        -   each of R and R^(I) is independently hydrogen or alkyl;    -   C is a ligand other than A or B; and    -   X is a counter ion,

wherein the number of coordinating atoms is 6) with the exception of[Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂.

The ligand A may be selected from the group consisting of NCS,imidazole, pyrazole, thiazole, oxazole, isoquinoline, substitutedpyridyl (eg 3- and/or 4-substituted pyridyl) and isomers thereof.

The ligand A may be selected from the group consisting of imidazole,pyrazole, thiazole, oxazole and isomers thereof.

The ligand A may be or contain a 5- or 6-membered aromatic ligandcontaining 3 nitrogen heteroatoms. The ligand A is preferably triazineor triazole.

The ligand A may be guanine or adenine.

The ligand A may be substituted by one or more substituents selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl,C₂-C₆ alkynyl, halogen, carboxy, amino, C₁₋₆-alkylamino,C₁₋₆-dialkylamino and hydroxyl.

The ligand A may be substituted by one or more substituents selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl,C₂-C₆ alkynyl and halogen. Preferably the ligand A is substituted by oneor more substituents selected from the group consisting of methyl,ethyl, propyl, iso-propyl, butyl, t-butyl, methoxy, ethoxy, ethenyl,propenyl, butenyl, ethynyl and propynyl.

The ligand B may be a bi-, tri- or tetra-dentate ligand which may belinear having the formula R¹RN(C₂H₄NR)_(r)R¹ or cyclic having theformula (RNC₂H₄)_(v), (RNC₂H₄)_(p)(RNC₃H₆)_(q) or[(RNC₂H₄)(RNC₃H₆)]_(s), wherein r is an integer selected from theintegers 1-3, v is 3 or 4, each of p and q is an integer independentlyselected from the integers 1-3 whereby the sum of p and q is 4 and s is2 or 3.

The ligand B is preferably a tri- or tetra-dentate ligand which iscyclic having the formula (RNC₂H₄)_(v), wherein v is 3 or 4.

The ligand B may be 1,4,7-trimethyl-1,4,7-triazacyclononane,1,4,8,11-tetramethyl-1,4,8,11-tetra-azacyclotetradecane,1,1,4,7,10,10-hexamethyltriethylenetetramine,1,2-dimethylethylenediamine or 1,1,2,2-tetramethylethylenediamine.

The ligand B may be 1,4,7-trimethyl-1,4,7-triazacyclononane,1,1,4,7,10,10-hexamethyltriethylenetetramine,1,2-dimethylethylenediamine or 1,1,2,2-tetramethylethylenediamine.

A preferred ligand B is 1,4,7-trimethyl-1,4,7-triazacyclononane.

The ligand C may be selected from the group consisting of an amineligand (such as NH₃ or NMe₃), CO, CN, a halogen, acetylacetonate (acac),3-bromo-acetylacetonate (Bracac), oxalate, tropolone, 1,4,7-triethylenecrown ether and 5-chloro-8-hydroxyquinoline.

A preferred ligand C is acac.

The geometry of the complex of Formula I may be cis or trans when eachof ligands A, B and C is bi-dentate.

The oxidation state of the metal in the complex of Formula I may be 2+or 3+. The oxidation state of the metal in the complex of Formula I ispreferably 3+.

The ligands A and B may be selected such that the overall charge on thecomplex of Formula I is selected from the group consisting of +3, +2,+1, 0, −1, −2 and −3.

The counterion X may be, F⁻, Cl⁻, Br⁻, I⁻, NO₃ ⁻, NR₄ ⁺, NR₄ ⁺, PF₆ ⁻,CF₃SO₃ ⁻, SO₄ ²⁻, ClO₄ ⁻, K⁺, Na⁺ Li⁺ or a combination thereof.

In the fourth aspect of the invention, the complex of Formula I may be[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-methylpyridine)]Cl₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3-chloropyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(isonicotinamide)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)pyrazine](NO3)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-methoxypyridine)](NO₃)₂,Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-dimethylaminopyridine))(NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-t-butyl-pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(isoquinoline)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(tropolone)(pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(tropolone)(4-t-butyl-pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3,4-dimethylpyridine)](CF₃SO₃)₂or[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3-hydroxypyridine)](NO₃)₂.

In the fourth aspect of the invention, the complex of Formula I may be[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](PF₆)₂or[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](NO₃)₂.

In the complex of Formula I, the metal may be selected to be rutheniumor osmium as desired. A person skilled in the art will appreciate thatsubstituting Ru with Os will change the working potential of a complexby around +400 mV to +600 mV and that the working potential may befurther fine tuned (in the reverse direction if necessary) by alteringthe ligands around the metal centre until the mediator reaches a workingpotential of −300 mV to +300 mV vs Ag/AgCl.

The term “alkyl” used herein includes linear or branched, saturatedaliphatic hydrocarbons. Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, tert-butyl and cyclopentyl. Unlessotherwise noted, the term “alkyl” includes alkyl and cycloalkyl groups.

The term “alkoxy” used herein describes an alkyl group joined to theremainder of the structure by an oxygen atom. Examples of alkoxy groupsinclude methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, tert-butoxy andcyclopentoxy. Unless otherwise noted, the term “alkoxy” includes alkoxyand cycloalkoxy groups.

The term “alkenyl” used herein describes an unsaturated, linear orbranched aliphatic hydrocarbon having at least one carbon-carbon doublebond. Examples of alkenyl groups include ethenyl, 1-propenyl,2-propenyl, 1-butenyl, 2-methyl-1-propenyl and cyclopentenyl. Unlessotherwise noted, the term “alkenyl” includes alkenyl and cycloalkenylgroups.

The term “acac” refers to the acetylacetonate anion which is theconjugate base of 2,4-pentanedione.

A “substituted” functional group (eg substituted alkyl, alkenyl, oralkoxy group) includes at least one substituent selected from thefollowing: halogen, alkoxy, mercapto, aryl, alkoxycarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, —OH, —NH₂, alkylamino,dialkylamino, trialkylammonium, alkanoylamino, dialkanoylamino,arylcarboxamido, hydrazino, alkylthio, alkenyl and reactive groups.

A “reactive group” is a functional group of a molecule that is capableof reacting with another compound to couple at least a portion of thatother compound to the molecule. Reactive groups include carboxy,activated ester, sulfonyl halide, sulfonate ester, isocyanate,isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amino,acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine,alkyl halide, imidazole, pyridine, phenol, alkyl, sulfonate,halotriazine, imido ester, maleimide, hydrazide, hydroxy, andphoto-reactive azido aryl groups. As understood in the art, activatedesters generally include esters of succinimidyl, benzotriazolyl or arylsubstituted by electron withdrawing groups such as sulfo, nitro, cyano,or halo.

A “biological fluid” is a bodily fluid or bodily fluid derivative inwhich an analyte can be measured (eg blood, interstitial fluid, plasma,dermal fluid, sweat, saliva and tears).

An “electrochemical sensor” is a device configured to detect thepresence of or measure the concentration or amount of an analyte in asample via electrochemical oxidation or reduction reactions. Thesereactions typically can be transduced to an electrical signal that canbe correlated to an amount or concentration of analyte.

A “redox mediator” is an electron transfer agent for carrying electronsbetween an analyte or an analyte-reduced or analyte-oxidized enzyme andan electrode directly or via one or more additional electron transferagents.

The electrochemical cell may be a two-electrode, a three-electrode, afour-electrode or a multiple-electrode system. A two-electrode systemcomprises a working electrode and a pseudo reference electrode. Athree-electrode system comprises a working electrode, an ideal or pseudoreference electrode and a separate counter electrode. As used herein, apseudo reference electrode is an electrode that is capable of providinga substantially stable reference potential. In a two-electrode system,the pseudo reference electrode also acts as the counter electrode inthis case a current passes through it without substantially perturbingthe reference potential. As used herein, an ideal reference electrode isan ideal non-polarisable electrode through which no current passes.

The term “measurable signal” means a signal which can be readilymeasured (such as electrode potential, fluorescence, spectroscopicabsorption, luminescence, light scattering, NMR, IR, mass spectroscopy,heat change or a piezo-electric change).

The term “biochemical analyte” includes any measurable chemical orbiochemical substance that may be present in ‘a biological fluid (suchas an enzyme, an antibody, a DNA sequence or a microorganism).

In accordance with the present invention, monodentate and bidentate havetheir generally accepted meaning in the art ie a monodentate ligand is achemical moiety or group that has one potential coordinating atom. Amultidentate ligand is a chemical moiety or group that has more than onepotential coordinating atom. The number of potential coordinating atomsis indicated by the prefix (eg bi or tri).

Embodiments of the present invention will now be described in anon-limitative sense only by way of the following examples and withreference to the accompanying Figures in which:

FIG. 1: ESI mass spectrum (+ve mode) of[Ru^(II)(Me₃tacn)(acac)(1-MeIm)](PF₆) in acetone with the simulatedisotopic patterns;

FIG. 2: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂, 0.1 M KCl and 0.1 M TRIS buffer(pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 3: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂ and 1 mM NADH in the absence(black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 4: Calibration plot of oxidation current versus NADH concentrationfor a 10 mM [Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂ solution containing2.5 mg ml⁻¹ PdR. Currents were recorded after an oxidation potential of+0.15 V (vs Ag/AgCl reference) was applied to the working electrode on astandard Oxford biosensors screen printed carbon micro-electrode stripusing an Autolab PGSTAT12 potentiostat/galvanostat (Eco Chemie,Netherlands) connected to a multiplexer (MX452 Sternhagen design)controlled by the General Purpose Electrochemical System software (EcoChemie, Netherlands);

FIG. 5: Plasma Total cholesterol (TC) concentration versus oxidationcurrent recorded for a total cholesterol assay containing 80 mM[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂;

FIG. 6: Plasma triglycerides (TRG) concentration versus oxidationcurrent recorded for a triglyceride assay containing 80 mM[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂;

FIG. 7: Plasma high density lipoprotein (HDL) concentration versusoxidation current recorded for a HDL assay containing 40 mM[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂, 6 mg ml-1 TNAD, 4 mg ml-1 PdR,22 mg ml-1 cholesterol dehydrogenase, 23 mg ml-1 lipase, 2% wt/v BSA,0.1M sucrose monocaprate and 0.1M neomycin;

FIG. 8: ESI mass spectra (+ve mode) of [Ru^(II)(Me₃tacn)(acac)(py)]PF₆in CH₃CN;

FIG. 9: ESI mass spectra (+ve mode) of[Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂ in methanol;

FIG. 10: Cyclic voltammogram of [Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂ in abuffer solution containing NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M) (pH=8.05)in milli Q water. Glassy carbon as working electrode, platinum wire ascounter electrode, SCE as reference electrode. K₃[Fe(CN)₆] was used asinternal standard with +0.18 V vs SCE;

FIG. 11: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂, 0.1 M KCl and 0.1 M TRIS buffer (pH9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 12: UV absorbance spectroscopy of a solution consisting of 2 mM[Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂ and 5 mM NADH in the absence (black)and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 13: Calibration plot of oxidation current versus NADH concentrationfor a 10 mM [Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂ solution containing 1 mgmF⁻¹ PdR. Currents were recorded after an oxidation potential of +0.15 V(vs Ag/AgCl reference) was applied to the working electrode on astandard Oxford Biosensors screen printed carbon micro-electrode stripusing an Autolab PGSTAT12 potentiostat/galvanostat (Eco Chemie,Netherlands) connected to a multiplexer (MX452 Sternhagen design)controlled by the General Purpose Electrochemical System software (EcoChemie, Netherlands);

FIG. 14: Plasma total cholesterol calibration plot of oxidation currentfor a total cholesterol sensor containing 50 mM[Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂ and 3% Anameg-7, NAD(9.6 mg/ml),PdR(4.3 mg/ml), ChE(3.3 mg/ml), ChDh(42 mg/ml), Anameg-7(3%),myo-inositol (15 mg/ml), ectoine (15 mg/ml) in TRIS pH 9. Currents wererecorded after an oxidation potential of +0.15 V (vs Ag/AgCl reference)was applied to the working electrode on a standard Oxford Biosensorsscreen printed carbon micro-electrode strip using an Autolab PGSTAT12potentiostat/galvanostat (Eco Chemie, Netherlands) connected to amultiplexer (MX452, Sternhagen Design), controlled by the GeneralPurpose Electrochemical System software (Eco Chemie, Netherlands);

FIG. 15: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃TACN)(acac)(4-MePy)]Cl₂, 0.1 M KCl and 0.1 M TRIS buffer(pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 16: UV absorbance spectroscopy of a solution consisting of 1.25 mM[Ru^(III)(Me₃TACN)(acac)(4-MePy)]Cl₂ and 1.25 mM NADH in the absence(black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 17: Calibration plot of oxidation current versus the totalcholesterol (TC) concentration for different lyophilized serum samples.Currents were recorded after an oxidation potential of +0.15 V (vsAg/AgCl reference) was applied to the working electrode on a standardOxford Biosensors screen printed carbon micro-electrode strip using an.Autolab PGSTAT12 potentiostat/galvanostat (Eco Chemie, Netherlands)connected to a multiplexer (MX452 Sternhagen design) controlled by theGeneral Purpose Electrochemical System software (Eco Chemie,Netherlands);

FIG. 18: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃tacn)(acac)(3-Clpy)](NO₃)₂ in methanol (insets show theexpanded isotopic patterns). a) calculated isotopic patterns of[Ru^(III)(Me₃tacn)(acac)(3-Clpy)]²⁺ and b) calculated isotopic patternof [Ru^(II)(Me₃tacn)(acac)(3-Clpy)]⁺;

FIG. 19: Cyclic voltammogram of [Ru^(III)(Me₃tacn)(acac)(3-Clpy)](NO₃)₂in a buffer solution containing NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M)(pH=8.20) in milli Q water. Glassy carbon as working electrode, platinumwire as counter electrode, SCE as reference electrode. K₃[Fe(CN)₆] wasused as internal standard with +0.18 V vs SCE;

FIG. 20: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(acac)(3-Clpy)](NO₃)₂ and 1 mM NADH in the absence(black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 21: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃tacn)(acac)(3-Clpy)](NO₃)₂, 0.1 M KCl and 0.1 M TRIS buffer(pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 22: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru(Me₃tacn)(acac)(isna)](NO₃)₂, 0.1 M KCl and 0.1 M TRIS buffer (pH9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 23: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru(Me₃tacn)(acac)(isna)](NO₃)₂ and 1 mM NADH in the absence (black) andpresence (grey) of 0.066 mg ml⁻¹ PdR;

FIG. 24: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃TACN)(acac)(pz)](NO₃)₂, 0.1 M KCl and 0.1 M TRIS buffer (pH9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 25: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃TACN)(acac)(pz)](NO₃)₂ in acetone (insets show theexperimental and simulated isotopic patterns);

FIG. 26: Cyclic voltammogram of [Ru^(III)(Me₃tacn)(acac)(pz)](NO₃)₂ inbuffer solution containing NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M) (pH=8.20)in milli Q water. Glassy carbon as working electrode, platinum wire ascounter electrode, SCE as reference electrode. K₃[Fe(CN)₆] was used asinternal standard with +0.18 V vs SCE;

FIG. 27: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(acac)(pz)](NO₃)₂ and 1 mM NADH in the absence (black)and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 28: Calibration plot of oxidation current versus the totalcholesterol (TC) concentration for different lyophilized serum samples.Currents were recorded after an oxidation potential of +0.15 V (vsAg/AgCl reference) was applied to the working electrode on a standardOxford Biosensors screen printed carbon micro-electrode strip using anAutolab PGSTATI2 potentiostat/galvanostat (Eco Chemie, Netherlands)connected to a multiplexer (MX452 Sternhagen design) controlled by theGeneral Purpose Electrochemical System software (Eco Chemie,Netherlands);

FIG. 29: Cyclic voltammetry of [Ru^(III)(Me₃tacn)(acac)(3-MeO-py)](NO₃)₂recorded using a two electrode configuration with the standard OB wellelectrode as working electrode and an on-chip Ag/AgCl counter-referenceelectrode and with a scan rate of 100 mV s⁻¹. The black line is for 10mM [Ru^(III)(Me₃tacn)(acac)(3-MeO-py)](NO₃)₂ in 0.1 M Tris pH9 buffercontaining 0.1 M KCl and 10 mM NADH and the grey line is the samesolution after addition of 2.5 mg ml⁻¹ PdR;

FIG. 30: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃tacn)(acac)(4-OMe-py)](NO₃)₂ in methanol (insets show theexpanded isotopic patterns);

FIG. 31: Cyclic voltammogram of[Ru^(III)(Me₃tacn)(acac)(4-MeO-py)](NO₃)₂ in a buffer solution ofNaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M) (pH=8.20). Glassy carbon as workingelectrode, platinum wire as counter electrode, SCE as referenceelectrode. K₃[Fe(CN)₆] was used as internal standard (+0.18 V vs SCE);

FIG. 32: Cyclic voltammetry of[Ru^(III)(Me₃tacn)(acac)(3-OMe-py)](NO₃)₂, recorded using a twoelectrode configuration with the standard OB well electrode as workingelectrode and an on-chip Ag/AgCl counter-reference electrode and with ascan rate of 100 mV s⁻¹. The black line is for 10 mM[Ru^(III)(Me₃tacn)(acac)(4-OMe-py)](NO₃)₂ in 0.1 M Tris pH9 buffercontaining 0.1 M KCl and 10 mM NADH, and the grey line is the samesolution after addition of 2.5 mg ml⁻¹ PdR;

FIG. 33: Calibration plot of oxidation current versus NADH concentrationfor a 10 mM [Ru^(III)(Me₃tacn)(acac)(4-OMe-py)](NO₃)₂ solutioncontaining 2.5 mg ml⁻¹ PdR. Currents were recorded after an oxidationpotential of +0.15 V (vs Ag/AgCl reference) was applied to the workingelectrode on a standard Oxford Biosensors screen printed carbonmicro-electrode strip using an Autolab PGSTAT12 potentiostat/galvanostat(Eco Chemie, Netherlands) connected to a multiplexer (MX452 Sternhagendesign) controlled by the General Purpose Electrochemical Systemsoftware (Eco Chemie, Netherlands);

FIG. 34: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂ in methanol;

FIG. 35: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂, 0.1 M KCl and 0.1 M TRIS buffer(pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 36: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂ and 1 mM NADH in the absence(black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 37: Calibration plot of oxidation current versus NADH concentrationfor a 10 mM [Ru^(III)(Me₃tacn)(acac)(1-MeIm)](NO₃)₂ solution containing2.5 mg ml⁻¹ PdR. Currents were recorded after an oxidation potential of+0.15 V (vs Ag/AgCl reference) was applied to the working electrode on astandard Oxford Biosensors screen printed carbon micro-electrode stripusing an Autolab PGSTAT12 potentiostat/galvanostat (Eco Chemie,Netherlands) connected to a multiplexer (MX452 Sternhagen design)controlled by the General Purpose Electrochemical System software (EcoChemie, Netherlands);

FIG. 38: ESI mass spectrum of [Ru^(III)(Me₃tacn)(acac)(1-MeIm)](CF₃SO₃)₂in acetone (+ve mode);

FIG. 39: Cyclic voltammogram of[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](CF₃SO₃)₂ in a buffer solutioncontaining NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M) (pH=8.05) in H₂O Glassycarbon as working electrode, platinum wire as counter electrode, SCE asreference electrode. K₃[Fe(CN)₆] was used as internal standard with+0.18 V vs SCE;

FIG. 39 a: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃TACN)(acac)(1-MeIm)](CF₃SO₃)₂, 0.1 M KCl, 1% chaps and 0.1M TRIS buffer (pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 39 b: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃TACN)(acac)(1-MeIm)](CF₃SO₃)₂, 0.1 M KCl, 1% chaps, 0.1 MTRIS buffer (pH 9.0) and 1 mM NADH in the absence (black) and presence(grey) of 0.030 mg ml⁻¹ PdR;

FIG. 39 c: Calibration plot of oxidation current versus NADHconcentration for a 10 mM [Ru^(III)(Me₃TACN)(acac)(1-MeIm)](CF₃SO₃)₂solution containing 2.5 mg ml⁻¹ PdR. Currents were recorded after anoxidation potential of +0.15 V (vs Ag/AgCl reference) was applied to theworking electrode on a standard Oxford Biosensors screen printed carbonmicro-electrode strip using an Autolab PGSTAT12 potentiostatlgalvanostat(Eco Chemie, Netherlands) connected to a to a multiplexer (MX452,Sternhagen Design) controlled by the General Purpose ElectrochemicalSystem software (Eco Chemie, Netherlands);

FIG. 40: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃tacn)(acac)(4-Me₂N-py)](NO₃)₂.H₂O in methanol with theexperimental and simulated isotopic patterns;

FIG. 41: Cyclic voltamrriogram of[Ru^(III)(Me₃tacn)(acac)(4-Me₂N-py)](NO₃)₂.H2O in a buffer solutioncontaining NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M) (pH=8.20) in milli Qwater. Glassy carbon as working electrode, platinum wire as counterelectrode, SCE as reference electrode. K₃[Fe(CN)6] was used as internalstandard with +0.18 V vs SCE;

FIG. 42: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃tacn)(acac)(4-Me₂N-py)](NO₃)₂, 0.1 M KCl and 0.1 M TRISbuffer (pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 43: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(acac)(4-Me₂N-py)](NO₃)₂ and 1 mM NADH in the absence(black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 44: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)](NO₃)₂.3H₂O in methanol with theexperimental and simulated isotopic patterns;

FIG. 45: Cyclic voltammogram of[Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)](NO₃)₂.3H₂O in a buffer solutioncontaining NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M) (pH=8.20) in milli Qwater. Glassy carbon as working electrode, platinum wire as counterelectrode, SCE as reference electrode. K₃[Fe(CN)₆] was used as internalstandard with +0.18 V vs SCE;

FIG. 46: Cyclic voltammetry of 10 mM[Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)](NO₃)₂ in 0.1 M Tris pH9 bufferrecorded using a two electrode configuration with the standard OB wellelectrode as working electrode and an on-chip Ag/AgCl counter-referenceelectrode and with a scan rate of 100 mV s⁻¹;

FIG. 47: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)](NO₃)₂ and 1 mM NADH in the absence(black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 48: Calibration plot of oxidation current versus NADH concentrationfor a 10 mM [Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)](NO₃)₂ solutioncontaining 2.5 mg ml⁻¹ PdR. Currents were recorded after an oxidationpotential of +0.15 V (vs Ag/AgCl reference) was applied to the workingelectrode on a standard Oxford Biosensors screen printed carbonmicro-electrode strip using an Autolab PGSTAT12 potentiostatlgalvanostat(Eco Chemie, Netherlands) connected to a multiplexer (MX452 Sternhagendesign) controlled by the General Purpose Electrochemical Systemsoftware (Eco Chemie, Netherlands);

FIG. 49: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(acac)(isoquinoline)](NO₃)₂ and 1 mM NADH in theabsence (black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 50: Cyclic voltammetry of 10 mM[Ru^(III)(Me₃tacn)(acac)(isoquinoline)](NO₃)₂ in 0.1 M Tris pH9 buffer,recorded using a two electrode configuration with the standard OB wellelectrode as working electrode and an on-chip Ag/AgCl counter-referenceelectrode and with a scan rate of 100 mV s⁻¹;

FIG. 51: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃tacn)(tropolone)(pyridine)]PF₆ in acetone with experimentaland simulated isotopic patterns;

FIG. 52: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃tacn)(tropolone)(pyridine)](NO₃)₂ in methanol with theexperimental and simulated isotopic patterns;

FIG. 53: Cyclic voltammogram of [Ru^(III)(Me₃tacn)(tropolone)(py)](NO₃)₂in a buffer solution containing NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M)(pH=8.20) in milli Q water. Glassy carbon as working electrode, platinumwire as counter electrode, SCE as reference electrode. K₃[Fe(CN)₆] wasused as internal standard with +0.18 V vs. SCE;

FIG. 54: Calibration plot of oxidation current versus NADH concentrationfor a 10 mM [Ru^(III)(Me₃tacn)(tropolone)(py)](NO₃)₂ solution containing2.5 mg ml⁻¹ PdR. Currents were recorded after an oxidation potential of+0.15 V (vs Ag/AgCl reference) was applied to the working electrode on astandard Oxford Biosensors screen printed carbon micro-electrode stripusing an Autolab PGSTAT12 potentiostat/galvanostat (Eco Chemie,Netherlands) connected to a multiplexer (MX452 Sternhagen design)controlled by the General Purpose Electrochemical System software (EcoChemie, Netherlands);

FIG. 55: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(tropolone)(py)](NO₃)₂ and 1 mM NADH in the absence(black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 56: Cyclic voltammetry of 10mM[Ru^(III)(Me₃tacn)(tropolone)(py)](NO₃)₂ in 0.1 M Tris pH9 bufferrecorded using a two electrode configuration with the standard OB wellelectrode as working electrode and an on-chip Ag/AgCl counter-referenceelectrode and with a scan rate of 100 mV s⁻¹;

FIG. 57: ESI mass spectrum (+ve mode) of[Ru^(III)(Me₃tacn)(tropolone)(4-tert-butyl-py)](NO₃)₂ in methanol withthe experimental and simulated isotopic patterns;

FIG. 58: Cyclic voltammogram of[Ru^(III)(Me₃tacn)(tropolone)(4-tert-butyl-py)](NO₃)₂ in a buffersolution containing NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M) (pH=8.20) inmilli Q water. Glassy carbon as working electrode, platinum wire ascounter electrode, SCE as reference electrode. K₃[Fe(CN)₆] was used asinternal standard with +0.18 V vs. SCE;

FIG. 59: Cyclic voltammetry of 10 mM[Ru^(III)(Me₃tacn)(tropolone)(4-tert-butyl-py)](NO₃)₂ in 0.1 M Tris pH9buffer, recorded using a two electrode configuration with the standardOB well electrode as working electrode and an on-chip Ag/AgClcounter-reference electrode and with a scan rate of 100 mVs⁻¹;

FIG. 60: Calibration plot of oxidation current versus NADH concentrationfor a 10 mM [Ru^(III)(Me₃tacn)(tropolone)(4-tert-butyl-py)](NO₃)₂solution containing 1 mg ml⁻¹ PdR. Currents were recorded after anoxidation potential of +0.15 V (vs Ag/AgCl reference) was applied to theworking electrode on a standard Oxford Biosensors screen printed carbonmicro-electrode strip using an Autolab PGSTATI2 potentiostat/galvanostat(Eco Chemie, Netherlands) connected to a multiplexer (MX452 Sternhagendesign) controlled by the General Purpose Electrochemical Systemsoftware (Eco Chemie, Netherlands);

FIG. 61: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃tacn)(tropolone)(4-tert-butyl-py)](NO₃)₂ and 1 mM NADH inthe absence (black) and presence (grey) of 0.033 mg ml⁻¹ PdR;

FIG. 62: ESI mass spectrum of[Ru^(III)(Me₃tacn)(acac)(3,4-Me₂-py)](CF₃SO₃) in acetone (+ve mode);

FIG. 63: Cyclic voltammogram of[Ru^(III)(Me₃tacn)(acac)(3,4-Me₂py)](CF₃SO₃)₂ in buffer solutioncontaining NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M) (pH=8.05) in H₂O Glassycarbon as working electrode, platinum wire as counter electrode, SCE asreference electrode. K₃[Fe(CN)₆] was used as internal standard with+0.18 V vs. SCE;

FIG. 63 a: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃TACN)(acac)(3,4-Me₂py)](CF₃SO₃)₂, 0.1 M KCl, 1% chaps and0.1 M TRIS buffer (pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 63 b: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃TACN)(acac)(3,4-Me₂py)](CF₃SO₃)₂, 0.1 M KCl, 1% chaps, 0.1M TRIS buffer (pH 9.0) and 1 mM NADH in the absence (black) and presence(grey) of 0.030 mg ml⁻¹ PdR;

FIG. 63 c: Calibration plot of oxidation current versus NADHconcentration for a 10 mM [Ru^(III)(Me₃TACN)(acac)(3,4-Me₂py)](CF₃SO3)₂solution containing 2.5 mg ml-1 PdR. Currents were recorded after anoxidation potential of +0.15 V (vs Ag/AgCl reference) was applied to theworking electrode on a standard Oxford Biosensors screen printed carbonmicro-electrode strip using an Autolab PGSTATI2 potentiostatlgalvanostat(Eco Chemie, Netherlands) connected to a to a multiplexer (MX452,Sternhagen Design) controlled by the General Purpose ElectrochemicalSystem software (Eco Chemie, Netherlands);

FIG. 64: ESI-MS of [Ru^(III)(Me₃tacn)(acac)(3-OHpy)](NO₃)₂ (+ve mode) inmethanol. (Insets show the experimental (top) and simulated (bottom)isotopic patterns;

FIG. 65: Cyclic voltammogram of [Ru^(III)(Me₃tacn)(acac)(3-OHpy)](NO₃)₂in buffer solution containing NaH₂PO₄ (0.005 M)/Na₂HPO₄ (0.094 M)(pH=8.05) in H₂O Glassy carbon as working electrode, platinum wire ascounter electrode, SCE as reference electrode. K₃[Fe(CN)₆] was used asinternal standard with +0.18 V vs. SCE;

FIG. 65 a: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 10 mM[Ru^(III)(Me₃TACN)(acac)(3-OHpy)](NO₃)₂, 0.1 M KCl, 1% chaps and 0.1 MTRIS buffer (pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 65 b: UV absorbance spectroscopy of a solution consisting of 1 mM[Ru^(III)(Me₃TACN)(acac)(3-OHpy)](NO₃)₂, 0.1 M KCl, 1% chaps, 0.1 M TRISbuffer (pH 9.0) and 1 mM NADH in the absence (black) and presence (grey)of 0.030 mg ml⁻¹ PdR;

FIG. 65 c: Calibration plot of oxidation current versus NADHconcentration for a 10 mM [Ru^(III)(Me₃TACN)(acac)(3-OHpy)](NO₃)₂solution containing 2.5 mg ml-1 PdR. Currents were recorded after anoxidation potential of +0.15 V (vs Ag/AgCl reference) was applied to theworking electrode on a standard Oxford Biosensors screen printed carbonmicro-electrode strip using an Autolab PGSTAT12 potentiostat/galvanostat(Eco Chemie, Netherlands) connected to a multiplexer (MX452, SternhagenDesign) controlled by the General Purpose Electrochemical Systemsoftware (Eco Chemie, Netherlands);

FIG. 66: ESI mass spectrum of [Ru^(III)(tmc)(NCS)₂](ClO₄) in methanolwith the isotopic pattern;

FIG. 67: Cyclic voltammogram of [Ru^(III)(tmc)(NCS)₂](ClO₄) in 0.1 MTFA. Glassy carbon as working electrode, platinum wire as counterelectrode, SCE as reference electrode. K₃[Fe(CN)₆] was used as internalstandard with +0.18 V vs. SCE;

FIG. 67 a: Cyclic voltammogram for a standard Oxford Biosensors screenprinted carbon micro-electrode strip in a solution consisting of 0.32 mM[Ru^(III)(TMC)(NCS)₂](ClO₄), 0.1 M KCl, 1% chaps and 0.1 M TRIS buffer(pH 9.0) recorded with a scan rate of 100 mVs⁻¹;

FIG. 67 b: UV absorbance spectroscopy of a solution consisting of 0.01mM [Ru^(III)(TMC)(NCS)₂](ClO₄), 0.1 M KCl, 1% chaps, 0.1 M TRIS buffer(pH 9.0) and 1 mM NADH in the absence (black) and presence (grey) of0.030 mg ml⁻¹ PdR;

FIG. 67 c: Calibration plot of oxidation current versus NADHconcentration for a 0.32 mM [Ru^(III)(TMC)(NCS)₂](ClO₄) solutioncontaining 2.5 mg ml⁻¹ PdR. Currents were recorded after an oxidationpotential of +0.15 V (vs Ag/AgCl reference) was applied to the workingelectrode on a standard Oxford Biosensors screen printed carbonmicro-electrode strip using an Autolab PGSTATI2 potentiostat/galvanostat(Eco Chemie, Netherlands) connected to a multiplexer (MX452, SternhagenDesign) controlled by the General Purpose Electrochemical Systemsoftware (Eco Chemie, Netherlands);

FIG. 68: Sensor responses plotted vs. time for each CK (see Example 17);

FIG. 69: Sensor responses plotted vs creatinine concentration (seeExample 18);

FIG. 70: Sensor responses plotted vs glucose concentration (see Example19); and

FIG. 71: [Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂ glucose mix testingsaliva with standard additions of glucose.

Throughout the Examples which follow L denotes the ligand1,4,7-trimethyl-1,4,7-triazacyclononane. The material[Ru^(II)(L)(acac)(OH)]PF₆ is prepared according to Schneider et al:Inorg. Chem., 1993, 32, 4925. All of the Examples use 0.1M KCl and 1%CHAPS.

EXAMPLE 1 See FIGS. 1 to 7 [Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂

Method 1 Preparation of [Ru^(II)(Me₃TACN)(acac)(1-MeIm)]PF₆

N-methylimidazole (0.5 g, 6.0 mmol) was added to[Ru^(II)(L)(acac)(OH)]PF₆ (100 mg, 0.19 mmol) in absolute ethanol (5mL). The solution was refluxed under argon in the presence of a fewpieces of Zn amalgam for 24 h. After cooling to room temperature,acetone (15 mL) was added and the solution was then filtered. Thefiltrate was evaporated to dryness to give an orange solid which wasfiltered and washed with diethyl ether. Yield (100 mg). ESI/MS (positivemode) in acetone: m/z=454.4, [M]⁺.

Preparation of [Ru^(III)(L)(acac)(1-MeIm)](NO₃)₂

A solution of AgCF₃SO₃ (45 mg, 0.17 mmol) in acetone (2 mL) was slowlyadded to [Ru^(II)(Me₃tacn)(acac)(1-MeIm)]PF₆ (100 mg, 0.17 mmol) inacetone (3 mL). After 3 minutes the purple solution was filtered toremove the silver. [^(n)Bu₄N]NO₃ (1500 mg, 0.5 mmol) was then added togive a purple precipitate which was filtered and washed with acetone.Yield (50 mg). ESI/MS (positive mode) in methanol: m/z=277.5, [M[²⁺.E_(1/2) of Ru^(III/II)=+0.09 V vs. NHE in buffer solution (pH 8.20).

EXAMPLE 2 See FIG. 8-14 [Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂

Preparation of [Ru^(II)(DMSO)₄Cl₂]

Ruthenium trichloride trihydrate (1.0 g) was refluxed in dimethylsulphoxide (5 mL) for 5 minutes The volume was reduced to half in vacuoand addition of acetone (20 mL) gave a yellow precipitate. The yellowcomplex which separated was filtered off, washed with acetone and etherand vacuum dried.

Preparation of [Ru^(III)(L)Cl₃]

To a mixture of Ru^(II)(DMSO)₄Cl₂ (1.0 g, 2.1 mmol) in absolute ethanol(25 mL) was added L (0.80 g, 4.7 mmol) with stirring. The suspension washeated to 60° C. for 1 h until a clear deep red-brown solution wasobtained which was then refluxed for 2 h. The solvent was removed underreduced pressure by rotary evaporation. The red-orange residue wastreated with concentrated HCl and heated under reflux for 30 min in thepresence of air. An orange microcrystalline solid was collected byfiltration, washed with H₂O, ethanol and diethyl ether and air-dried.

Preparation of [Ru^(III)(L)(acac)(OH)]PF₆.H₂O

Solid Ru^(III)(L)Cl₃ (2.0 g; 5.0 mmol) was added in small amounts to asolution of sodium 2,4-pentanedionate (acac) (3.0 g; ˜24 mmol) in water(60 mL) with stirring at ambient temperature. The mixture was stirredfor 3.5 h until a clear red solution was obtained. Addition of asolution of NaPF₆ (2.0 g) in H₂O (5 mL) and cooling to 0° C. initiatedthe precipitation of orange microcrystals which were collected byfiltration, washed with diethyl ether and air-dried.

Preparation of [Ru^(II)(L)(acac)(py)]PF₆

A solution containing [Ru^(II)(L)(acac)(OH)]PF₆ (105 mg, 0.20 mmol) inabsolute ethanol/pyridine (5 mL) (4:1, v/v) was heated to reflux underargon atmosphere in the presence of 10 pieces of Zn amalgram for 4 h.After cooling to ambient temperature, the red microcrystallineprecipitate was collected by filtration, washed with diethyl ether andair-dried. The product was recrystallized from acetone/diethyl ether.Yield: (94 mg, 79%) ESI/MS (positive mode): m/z=451, [M]⁺. E_(1/2) ofRu^(III/II)=−0.18 V vs. Fc^(+/0) in 0.1 M TBAH in CH₃CN.

Preparation of [Ru^(III)(L)(acac)(py)](NO₃)₂

A solution of AgCF₃SO₃ (42 mg, 0.16 mmol) in acetone (1 mL) was slowlyadded to an orange acetone solution (3 mL) containing[Ru^(II)(Me₃tacn)(acac)(py)]PF₆ (90 mg, 0.15 mmol). After stirring for 5minutes, solid [^(n)Bu₄N]NO₃ (304 mg, 1 mmol) was added and the purpleprecipitate was filtered, washed with acetone and then diethyl ether.The product was recrystallized from methanol/diethyl ether. Yield: (64mg, 87%) ESI/MS (positive mode): m/z=451.0, [M]⁺; 225.4, [M]²⁺. E_(1/2)of Ru^(III/II)=0.2 V vs. NHE in buffer solution (pH 8.05).

EXAMPLE 3 See FIGS. 15-17 [Ru(III)(Me₃TACN)(acac)(4-MePy)]Cl₂

Preparation of [Ru^(II)(Me₃tacn)(acac)(4-Mepy)]PF₆

4-picoline (0.4 g, 4 mmol) was added to [Ru^(III)(Me₃tacn)(acac)(OH)]PF₆(200 mg, 0.37 mmol) in absolute ethanol (15 mL). The solution wasrefluxed under argon in the presence of 20 pieces of Zn amalgam for 24h. After cooling to room temperature, the solution was filtered and thefiltrate was then evaporated to dryness to give a brown solid which wasfiltered and then washed with diethyl ether. Yield (290 mg). ESI/MS(positive mode) in acetone: m/z=465.2, [M]⁺. Anal. calcd. forC₂₀H₃₅N₄O₂PF₅Ru: C, 39.41; H, 5.79; N, 9.11. Found: C, 39.53; H, 5.82;N, 8.98.

Preparation of [Ru^(III)(Me₃tacn)(acac)(4-Mepy)](PF₆)₂

A solution of (NH₄)₂[Ce(NO₃)₆] (134 mg, 0.24 mmol) in acetone (10 mL)was slowly added to the orange solution in acetone (5 mL) containing[Ru^(II)(Me₃tacn)(acac)(4-Mepy)]PF₆ (120 mg, 0.20 mmol). After 3 minutesthe purple solid was filtered and washed with acetone. The purple solidwas then dissolved in deionized water (10 mL) and the solution wasfiltered, and NH₄PF₆ (133 mg, 0.82 mmol) was added to give a purpleprecipitate, which was filtered and washed with deionized water. Yield(105 mg). ESI/MS (positive mode) in acetone: m/z=232.8, [M]²⁺. Anal.calcd. for C₂₀H₃₅N₄O₂P₂F₁₂Ru: C, 31.84; H, 4.68; N, 7.43. Found: C,31.90; H, 4.65; N, 7.32.

Preparation of [Ru^(III)(Me₃tacn)(acac)(4-Mepy)l(NO₃)₂.H₂O

A solution of [^(n)Bu₄N]NO₃ (230 mg, 0.76 mmol) in acetone (5 mL) wasslowly added to the purple solution of[Ru^(III)(Me₃tacn)(acac)(4-Mepy)](PF₆)₂ (140 mg, 0.19 mmol) in acetone(10 mL). The purple precipitate was filtered, washed with acetone andvacuum dried. Yield (40 mg). ESI/MS (positive mode) in methanol:m/z=232.8, [M]²⁺. E_(1/2) of Ru^(III/II)=+0.18 V vs. NHE in buffersolution (pH 8.20). Anal. calcd. for C₂₀H₃₅N₆O₈Ru.H₂O: C, 39.60; H,6.15; N, 13.85. Found: C, 39.72; H, 6.01; N, 13.90.

EXAMPLE 4 See FIGS. 18-21 [Ru(III)(Me₃TACN)(acac)(3-Clpy)](NO₃)₂

Preparation of [Ru^(II)(L)(acac)(3-Clpy)]PF₆

A solution containing [Ru^(II)(L)(acac)(OH)]PF₆ (150 mg, 0.28 mmol) inabsolute ethanol/3-chloropyridine (5 mL) (4:1, v/v) was refluxed underargon in the presence of 10 pieces of Zn amalgam for 24 h. After coolingto room temperature, 15 mL acetone was added and the solution was thenfiltered. The filtrate was evaporated to dryness to give a brown solidwhich was filtered and then washed with diethyl ether. ESI/MS (positivemode): m/z=485.3, [M]⁺. Yield: (110 mg)

Preparation of [Ru^(III)(L)(acac)(3-Clpy)](NO₃)₂

A solution of AgCF₃SO₃ (45 mg, 0.17 mmol) in acetone (2 mL) was slowlyadded to the brown solution in acetone (3 mL) containing[Ru^(II)(Me₃tacn)(acac)(3-Clpy)]PF₆ (110 mg, 0.17 mmol). After 3 minutesthe purple solution was filtered to remove the silver and then[^(n)Bu₄N]NO₃ (300 mg, 1 mmol) was added to give a purple precipitatewhich was filtered and washed with acetone. ESI/MS (positive mode) inmethanol: m/z=485.3, [M]⁺; 242.9, [M]²⁺. E_(1/2) of Ru^(III/II)=+0.27 Vvs. NHE in buffer solution (pH 8.20).

EXAMPLE 5 See FIGS. 22-23 [Ru^(III)(Me₃tacn)(acac)(isna)](NO₃)₂

Preparation of [Ru^(II)(L)(acac)(ISNA)]PF₆(ISNA=isonicotinamide)

Solid isonicotinamide (1 g, 8.20 mmol) was added to a suspension of[Ru^(III)(L)(acac)(OH)]PF₆ (100 mg, 0.19 mmol) in absolute ethanol (5mL). The mixture was refluxed in the presence of a few pieces of Znamalgam for 24 h under argon. After cooling to room temperature, 15 mLacetone was added and the solution was then filtered. The filtrate wasevaporated to dryness to give a brown solid which was filtered and thenwashed with diethyl ether. Yield: 100 mg. The crude product was used fornext step without further purification.

Preparation of [Ru^(III)(L)(acac)(ISNA)](NO₃)₂

A solution of AgCF₃SO₃ (45 mg, 0.17 mmol) in acetone (2 mL) was slowlyadded with stirring to a brown solution of [Ru^(II)(L)(acac)(ISNA)]PF₆(100 mg) in acetone (3 mL). After 5 minutes the purple solution wasfiltered and then concentrated to ca. 0.5 mL followed by addition ofdiethyl ether (30 mL). The resulting purple solid was filtered andredissolved in acetonitrile (5 mL). Addition of [^(n)Bu₄N]NO₃ (300 mg, 1mmol) in acetonitrile (2 mL) gave dark purple crystals after standingfor 1 day. ESI/MS (positive mode) in methanol: m/z=494.4, [M]⁺; 247.3,[M]²⁺. E_(1/2) of Ru^(III/II)=+0.28 V vs. NHE in buffer solution (pH8.20).

EXAMPLE 6 See FIGS. 24-28 [Ru^(III)(Me₃TACN)(acac)Pz](NO₃)₂

Preparation of [Ru^(II)(L)(acac)(pz)]PF₆

Pyrazole (0.5 g, 7.3 mmol) was added to [Ru^(II)(L)(acac)(OH)]PF₆ (150mg, 0.28 mmol) in absolute ethanol (5 mL). The solution was refluxedunder argon in the presence of 10 pieces of Zn amalgam for 24 h. Aftercooling to room temperature, acetone (15 mL) was added and the solutionwas then filtered. The filtrate was evaporated to dryness to give anorange solid which was filtered and then washed with diethyl ether.Yield (130 mg).

Preparation of [Ru^(III)(L)(acac)(pz)](NO₃)₂

A solution of AgCF₃SO₃ (60 mg, 0.23 mmol) in acetone (3 mL) was slowlyadded to the orange solution in acetone (7 mL) containing[Ru^(II)(Me₃tacn)(acac)(pz)]PF₆ (130 mg, 0.22 mmol). After 3 minutes thepurple solution was filtered to remove silver. [^(n)Bu₄N]NO₃ (0.3 g,0.98 mmol) was then added to give a purple precipitate which wasfiltered and washed with acetone. Yield (100 mg). ESI/MS (positive mode)in methanol: m/z=439.5, [M-H]⁺. E_(1/2) of Ru^(III/II)=+0.14 V vs. NHEin buffer solution (pH 8.20).

EXAMPLE 7 See FIG. 29-33 [Ru^(III)(Me₃TACN)(acac)(4-MeO-py)](NO₃)₂

Preparation of [Ru^(II)(L)(acac)(4-MeO-py)]PF₆(1)

4-Methoxypyridine (0.4 g, 3.7 mmol) was added to[Ru^(II)(L)(acac)(OH)]PF₆ (120 mg, 0.22 mmol) in absolute ethanol (5mL). The solution was refluxed under argon in the presence of 10 piecesof Zn amalgam for 24 h. After cooling to room temperature, acetone (15mL) was added and the solution was then filtered. The filtrate wasevaporated to dryness to give an orange solid which was filtered andwashed with diethyl ether. Yield: (100 mg)

Preparation of [Ru^(III)(L)(acac)(4-MeO-py)](NO₃)₂

A solution of AgCF₃SO₃ (45 mg, 0.17 mmol) in acetone (5 mL) was slowlyadded to 1 (100 mg, 0.16 mmol) in acetone (5 mL). After 3 minutes thepurple solution was filtered and then concentrated to ca. 1 mL. Additionof Et₂O (30 mL) gave a purple solid which was filtered and washed withEt₂O. The purple solid was redissolved in acetone (5 mL). [^(n)Bu₄N]NO₃(300 mg, 1 mmol) was then slowly added. The resulting purple precipitatewas filtered and washed with acetone. ESI/MS (positive mode) inmethanol: m/z=240.8, [M]²⁺. E_(1/2) of Ru^(III/II)=+0.16 V vs. NHE inbuffer solution (pH 8.20).

EXAMPLE 8 See FIGS. 34-37 [Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂

Method 2 Preparation of [Ru^(III)(L)(acac)(1-MeIm)](PF₆)₂

NH₄PF₆ (200 mg, 1.27 mmol) and 1-methylimidazole (200 mg, 2.44 mmol)were added to a solution containing [Ru^(II)(L)(acac)(OH)]PF₆ (150 mg,0.28 mmol) in absolute ethanol (5 mL). The mixture was refluxed for 1 h.After cooling to room temperature, the dark purple solid was filtered,washed with ethanol (3×5 mL) and then air-dried. Yield: (160 mg). ESI/MS(positive mode) in acetone: m/z=227.4 [M]²⁺.

Preparation of [Ru^(III)(L)(acac)(1-MeIm)](NO₃)₂

A solution of [^(n)Bu₄N]NO₃ (200 mg, 0.67 mmol) in acetone (2 mL) wasslowly added to [Ru^(III)(L)(acac)(1-MeIm)](PF₆)₂ (100 mg, 0.13 mmol) inacetone (8 mL) and the mixture was allowed to stand for 30 minutes Theresulting purple precipitate was filtered, washed with acetone (3×5 mL)and then dried under vacuum. Yield: 70 mg. ESI/MS (positive mode) inmethanol: m/z=227.3 [M]²⁺. E_(1/2) of Ru^(III/II)=+0.07 V vs. NHE.UV-Vis (H₂O): λ_(max) [nm] (ε [mol⁻¹dm³cm⁻¹]) 289 (5175), 314sh (3920),583 (855).

EXAMPLE 8A See FIGS. 38-39 and 39 a-c[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](CF₃SO₃)₂ Preparation of[Ru^(III)(Me₃tacn)(acac)(1-MeIm)](CF₃SO₃)₂

To [Ru^(III)(Me₃tacn)(acac)(1-MeIm)](PF₆)₂ (200 mg, 0.27 mmol) dissolvedin a minimum amount of acetone was added neat triflic acid (0.5 mL) withvigorous stirring. The purple solution was then added dropwise todiethyl ether (400 mL). The purple precipitate was filtered and driedunder vacuum. Yield (100 mg). ESI/MS (positive mode) in methanol:m/z=277.5, [M]²⁺. E_(1/2) of Ru^(III/II)=+0.09 V vs. NHE in buffersolution (pH 8.20).

EXAMPLE 9 See FIGS. 40-43 [Ru(III)(Me₃-TACN)(acac)(4-Me₂N-py)](NO₃)₂

Preparation of [Ru^(II)(Me₃tacn)(acac)(4-Me₂N-py)]PF₆

4-Dimethylaminopyridine (0.3 g, 2.4 mmol) was added to[Ru^(III)(Me₃tacn)(acac)(OH)]PF₆ (150 mg, 0.28 mmol) in absolute ethanol(10 mL). The solution was refluxed under argon in the presence of 10pieces of Zn amalgam for 24 h. After cooling to room temperature, theorange solid was filtered and recrystallized from acetone/diethyl ether.Yield (120 mg). ESI/MS (positive mode) in acetone: m/z=494.1, [M]⁺.Anal. calcd. for C₂₁H₃₈N₅O₂PF₆Ru: C, 39.50; H, 6.00; N, 10.97. Found: C,39.73; H, 6.05; N, 10.81.

Preparation of [Ru^(III)(Me₃tacn)(acac)(4-Me₂N-py)](NO₃)₂.H₂O

A solution of AgCF₃SO₃ (50 mg, 0.19 mmol) in acetone (2 mL) was slowlyadded to the orange solution of [Ru^(II)(Me₃tacn)(acac)(4-Me₂N-py)]PF₆(120 mg, 0.19 mmol) in acetone (5 mL). After 5 minutes the purplesolution was filtered to remove the silver. [^(n)Bu₄N]NO₃ (150 mg, 0.5mmol) was then added to give a purple precipitate which was filtered,washed with acetone and vacuum dried. Yield (80 mg). ESI/MS (positivemode) in methanol: m/z=247.3, [M]²⁺. E_(1/2) of Ru^(III/II)=+0.07 V vs.NHE in buffer solution (pH 8.20). Anal. calcd. for C₂₁ H₃₈N₇O₈Ru.1H₂O:C, 39.68; H, 6.34; N, 15.42. Found: C, 39.95; H, 6.40; N, 15.61.

EXAMPLE 10 See FIGS. 44-48 [Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)](NO₃)₂

Preparation of [Ru^(II)(Me₃tacn)(acac)(4-^(t)Bupy)]PF₆

4-tert-butylpyridine (0.6 g, 4.1 mmol) was added to[Ru^(III)(Me₃tacn)(acac)(OH)]PF₆ (300 mg, 0.56 mmol) in absolute ethanol(45 mL). The solution was refluxed under argon in the presence of 30pieces of Zn amalgam for 24 h. After cooling to room temperature, thesolution was filtered and then evaporated to dryness. The brown solidwas filtered and washed with diethyl ether. Yield (290 mg). ESI/MS(positive mode) in acetone: m/z=507.3, [M]⁺. Anal. calcd. forC₂₃H₄₁N₄O₂PF₆Ru: C, 42.39; H, 6.34; N, 8.60. Found: C, 42.57; H, 6.40;N, 8.68.

Preparation of ]Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)](PF₆)₂

A solution of (NH₄)₂[Ce(NO₃)₆] (202 mg, 0.37 mmol) in acetone (10 mL)was slowly added to the orange solution in acetone (5 mL) containing[Ru^(II)(Me₃tacn)(acac)(4-tert-butylpyridine)]PF₆ (200 mg, 0.31 mmol).After 3 minutes the purple solid was filtered and washed with acetone.The purple solid was then dissolved in deionized water (10 mL). To thefiltered solution was added NH₄PF₆ (200 mg, 1.23 mmol) to give a purpleprecipitate which was filtered and washed with deionized water. Yield(150 mg). ESI/MS (positive mode) in acetone: m/z=253.7, [M]²⁺. Anal.calcd. for C₂₃H₄₁N₄O₂P₂F₁₂Ru: C, 34.68; H, 5.19; N, 7.03. Found: C,34.90; H, 5.14; N, 7.09.

Preparation of [Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)](NO₃)₂.3H₂O

A solution of [^(n)Bu₄N]NO₃ (183 mg, 0.60 mmol) in acetone (2 mL) wasslowly added to the purple solution of[Ru^(III)(Me₃tacn)(acac)(4-^(t)Bupy)RPF₆)₂ (120 mg, 0.15 mmol) inacetone (5 mL). The purple precipitate was filtered, washed with acetoneand then vacuum dried. Yield (65 mg). ESI/MS (positive mode) inmethanol: m/z=253.7, [M]²⁺. E_(1/2) of Ru^(III/II)=+0.18 V vs. NHE inbuffer solution (pH 8.20). Anal. calcd. for C₂₃H₄₁N₆O₈Ru.3H₂O: C, 40.34;H, 6.92; N, 12.27. Found: C, 40.42; H, 6.72; N, 12.29

EXAMPLE 11 See FIGS. 49 and 50[Ru^(III)(Me₃tacn)(acac)(isoquinoline)](NO₃)₂

Preparation of [Ru^(II)(Me₃tacn)(acac)(isoquinoline)]PF₆

Isoquinoline (0.4 g, 3.8 mmol) was added to[Ru^(III)(Me₃tacn)(acac)(OH)]PF₆ (200 mg, 0.38 mmol) in absolute ethanol(30 mL). The solution was refluxed under argon in the presence of 20pieces of Zn amalgam for 24 h. The resulting brown solution was cooledand then filtered. The filtrate was concentrated to ca. 1 mL. Diethylether was added and the brown precipitate was filtered, washed withdiethyl ether and then air dried. Yield (190 mg). ESI/MS (positive mode)in acetone: m/z=501.3, [M]⁺.

Preparation of [Ru^(III)(Me₃tacn)(acac)(isoquinoline)](PF₆)₂

A solution of (NH₄)₂[Ce(NO₃)₆] (194 mg, 0.35 mmol) in acetone (10 mL)was slowly added to the orange solution in acetone (5 mL) containing[Ru^(II)(Me₃tacn)(acac)(isoquinoline)]PF₆ (190 mg, 0.29 mmol). After 3minutes the purple solid was filtered and washed with acetone. Thepurple solid was then dissolved in 10 ml deionized water and thesolution was filtered. NH₄PF₆ (192 mg, 1.18 mmol) was added to give apurple precipitate which was filtered and washed with deionized water.Yield (120 mg). ESI/MS (positive mode) in acetone: m/z=250.7, [M]²⁺.

Preparation of [Ru^(III)(Me₃tacn)(acac)(isoquinoline)](NO₃)₂

A solution of [^(n)Bu₄N]NO₃ (274 mg, 0.90 mmol) in acetone (3 mL) wasslowly added to the purple solution of[Ru^(III)(Me₃tacn)(acac)(isoquinoline)](PF₆)₂ (240 mg, 0.30 mmol) inacetone (8 mL). The purple precipitate was filtered, washed with acetoneand vacuum dried. Yield (120 mg). ESI/MS (positive mode) in methanol:m/z=250.7, [M]²⁺. E_(1/2) of Ru^(III/II)=+0.21 V vs. NHE in buffersolution (pH 8.20).

EXAMPLE 12 See FIGS. 51-56 [Ru^(III)(Me₃tacn)(tropolone)(py)](NO₃)₂

Preparation of [Ru^(II)(Me₃tacn)(tropolone)(py)](PF₆)

A yellow mixture of [Ru^(III)(Me₃tacn)Cl₃] (120 mg, 0.32 mmol) andtropolone (47 mg, 0.38 mmol) in 5 mL H₂O was refluxed in air for 2 h.The resulting deep green solution was filtered and NH₄PF₆ (323 mg, 1.98mmol) was added to give a green precipitate which was filtered andwashed with deionized H₂O. The green solid was suspended in 10 mLethanol and pyridine (400 μL, 4.95 mmol) was added. The mixture was thenrefluxed under argon overnight in the presence of a few pieces of zincamalgam. The resulting brown solution was cooled and the brownprecipitate was filtered, washed with ethanol and then air dried. Yield:26%, 52 mg. ESI-MS: m/z=473.3, [M⁺].

Preparation of [Ru^(III)(Me₃tacn)(tropolone)(py)](NO₃)₂

A solution of AgCF₃SO₃ (32 mg, 0.12 mmol) in acetone (3 mL) was slowlyadded to the brown acetone solution (5 mL) of[Ru^(II)(Me₃tacn)(tropolone)(py)]PF₆ (52 mg, 0.08 mmol). The brownsolution turned green immediately and the mixture was stirred in thedark for 30 minutes. The silver metal in the solution was removed bycentrifuge and the green solution was then slowly added to ca. 80 mLdiethyl ether. The green precipitate was collected by filtration andwashed with diethyl ether. It was then dissolved in 5 mL acetone and asolution of [^(n)Bu₄N]NO₃ (77 mg, 0.25 mmol) in acetone (2 mL) wasslowly added. The green precipitate was filtered, washed with acetoneand vacuum dried. Yield: 86%, 41 mg. ESI-MS: m/z=236.8, [M²⁺]. E_(1/2)of Ru^(III/II)=0.25V vs. NHE in buffer solution (pH 8.20).

EXAMPLE 13 See FIGS. 57-61[Ru^(III)(Me₃tacn)(tropolone)(4-t-butyl-py)](NO₃)₂

Preparation of [Ru^(II)(Me₃tacn)(tropolone)(4-t-butyl-py)](PF₆)

A yellow mixture of [Ru^(III)(Me₃tacn)Cl₃] (120 mg, 0.32 mmol) andtropolone (47 mg, 0.38 mmol) in 5 mL H₂O was refluxed in air for 2 h.The resulting deep green solution was filtered and NH₄PF₆ (323 mg, 1.98mmol) was added to give a green precipitate which was filtered andwashed with deionized H₂O. The green solid was suspended in 10 mLethanol and 4-tert-butylpyridine (400 μL, 2.73 mmol) was added. Themixture was then refluxed under argon overnight in the presence of a fewpieces of zinc amalgam. The resulting brown solution was cooled and thebrown precipitate was filtered, washed with ethanol and then air dried.Yield: 93%, 200 mg. ESI-MS: m/z=529.3, [M⁺].

Preparation of [Ru^(III)(Me₃tacn)(tropolone)(4-t-butyl-py)](NO₃)₂

A solution of AgCF₃SO₃ (92 mg, 0.36 mmol) in acetone (5 mL) was slowlyadded to the brown acetone solution (10 mL) of[Ru^(II)(Me₃tacn)(tropolone)(4-t-butyl-py)]PF₆ (200 mg, 0.30 mmol). Thebrown solution turned green immediately and the mixture was stirred inthe dark for 30 minutes. The silver metal in the solution was removed bycentrifuge and the green solution was then slowly added to ca. 80 mLdiethyl ether. The green precipitate was collected by filtration andwashed with diethyl ether. It was then dissolved in 5 mL acetone and asolution of [^(n)Bu₄N]NO₃ (271 mg, 0.89 mmol) in acetone (5 mL) wasslowly added. The green precipitate was filtered, washed with acetoneand vacuum dried. Yield: 33%, 65 mg. ESI-MS: m/z=264.8, [M²⁺]. E_(1/2)of Ru^(III/II)=0.23V vs. NHE in buffer solution (pH 8.20).

EXAMPLE 14 See FIGS. 62-63 and 63 a-c[Ru^(III)(Me₃tacn)(acac)(3,4-Me₂py)](CF₃SO₃)₂

Preparation of [Ru^(II)(Me₃tacn)(acac)(3,4-Me₂py)](PF₆)

3,4-Lutidine (0.3 g, 2.8 mmol) was added to[Ru^(III)(Me₃tacn)(acac)(OH)]PF₆ (300 mg, 0.56 mmol) in absolute ethanol(10 mL). The solution was refluxed under argon in the presence Znamalgam (10 pieces) for 24 h. After cooling to room temperature, theorange solid was filtered and recrystallized from acetone/diethyl ether.Yield (300 mg).

Preparation of [Ru^(III)(Me₃tacn)(acac)(3,4-Me₂py)](CF₃SO₃)₂

A solution of AgCF₃SO₃ (135 mg, 0.48 mmol) in acetone (2 mL) was slowlyadded to an orange solution of [Ru^(II)(Me₃tacn)(acac)(3,4-Me₂py)](PF₆)(300 mg, 0.48 mmol) in acetone (10 mL). After 5 minutes the purplesolution was filtered and concentrated to ca 1 mL. Addition of diethylether (50 mL) gave a purple solid which was collected and recrystallizedfrom acetone/diethyl ether. Yield (300 mg). The purple solid (300 mg)was then re-dissolved in a minimum amount of acetone and neat triflicacid (0.5 mL) was then added with vigorous stirring. The purple solutionwas then slowly added to diethyl ether (500 mL). The purple precipitatewas filtered and dried under vacuum. Yield: (150 mg). ESI/MS (positivemode) in acetone: m/z=239.7, [M]²⁺. E_(1/2) of Ru^(III/II)=+0.17 V vs.NHE in buffer solution (pH 8.20).

EXAMPLE 15 See FIGS. 64-65 and 65 a-c[Ru^(III)(Me₃tacn)(acac)(3-OHpy)](NO₃)₂

Preparation of [Ru^(II)(Me₃tacn)(acac)(3-OHpy)](PF₆)

3-Hydroxypyridine (0.1 g, 1.05 mmol) was added to[Ru^(III)(Me₃tacn)(acac)(OH)]PF₆ (200 mg, 0.37 mmol) in absolute ethanol(10 mL). The solution was refluxed under argon in the presence Znamalgam (10 pieces) for 16 h. After cooling to room temperature, theorange solution was filtered and concentrated to ca. 1 mL. Addition ofdiethyl ether (50 mL) gave an orange solid which was filtered andrecrystallized from acetone/diethyl ether. Yield (160 mg).

Preparation of [Ru^(III)(Me₃tacn)(acac)(3-OHpy)](NO₃)₂

A solution of AgCF₃SO₃ (70 mg, 0.27 mmol) in acetone (2 mL) was slowlyadded to the orange solution of [Ru^(II)(Me₃tacn)(acac)(3-OHpy)](PF₆)(160 mg, 0.26 mmol) in acetone (8 mL). After 5 minutes the purplesolution was filtered and concentrated to ca 1 mL. Addition of diethylether (50 mL) gave a purple solid which was filtered and recrystallizedfrom acetone/diethyl ether. Yield (150 mg). The purple solid (150 mg)was then re-dissolved in acetone (8 ml) and a solution of[N^(n)Bu₄](NO₃) (200 mg) in acetone (2 ml) was then slowly added. Thepurple precipitate was filtered and dried under vacuum. Yield: (100 mg).ESI/MS (positive mode) in methanol: m/z=233.8, [M]²⁺. E_(1/2) ofRu^(III/II)=+0.14 V vs. NHE in buffer solution (pH 8.20).

EXAMPLE 16 See FIGS. 66-67 and 67 a-c [Ru^(III)(tmc)(NCS)₂](ClO₄)

Preparation of [Ru^(III)(TMC)(NCS)₂](ClO₄)

The purple solid is prepared according a literature method. (Che, C. M.;Kwong, S. S.; Poon C. K. Inorg. Chem. 1985, 24, 1601-1602).

EXAMPLE 17

The aim of the experiment was to demonstrate measurement of creatinekinase activity by wet testing with[Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂.

Enzyme Mixture

Enzyme mix was prepared with the following composition:

0.1M imidazole (balanced with acetic acid, pH 7.1 at 37° C.)

40 mM [Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂

20 mM nicotinamide adenine dinucleotide

5 mg/ml diaphorase

5 mg/ml glucose 6-phosphate dehydrogenase

20 mg/ml hexokinase

20 mM D-glucose

6.25 mM adenosine diphosphate (di-sodium salt)

30 mM magnesium acetate

5 mM EDTA (tetra sodium salt).

CK Solution

Lyophilized human recombinant CK sample was obtained from Asahi Kasei. Astock CK solution in buffer was made at 63.9 kU/L and diluted withbuffer to give samples with varying CK activity. The activities of theCK samples were determined using a Space clinical analyser(Schiappanelli Biosystems Inc).

Wet Testing Protocol

9.6 μL enzyme mix was placed in an eppendorf, to which was added 1.2 μLCK sample and 1.2 μL N-acetyl cysteine (200 mM). The eppendorf wasplaced on a heat block at 37° C. for 3 minutes to incubate the CK in thepresence of N-acetyl cysteine and hence activate the CK. The mix wasthen added to 1.2 μL creatine phosphate (1000 mM) at 37° C.

12 μl of a enzyme/CK mix was then immediately placed on the electrode,and the chronoamperometry test was initiated using a multiplexer (MX452,Sternhagen design) attached to an Autolab (PGSTAT 12).

The oxidation current was measured at 0.15 V at 15 time points (0, 14,28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182 and 196 seconds)with a reduction current measured at −0.45 V at the final time point(210 seconds). The transient current was measured for 1 second. Eachsample was tested in duplicate.

Analysis

The output from the GPES software was analysed using the DataAnal 2-17programme for converting data into a spreadsheet. These data were thentransferred to the data analysis template.

Results

The sensor responses were plotted vs. time for each CK sample (see FIG.68). The initial rate of response (change in current for the time period14-28 seconds) was determined for each sample and a plot made of rate(nA/min) vs. CK activity (kU/L). There was a linear dependence of therate of response on CK activity determined by the reference method.

EXAMPLE 18

The aim of the experiment was to demonstrate measurement of creatinineby wet testing with [Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂

Enzyme Mixture

Enzyme mix was prepared with the following composition:

0.1M Tris (balanced with HCl, pH 7.5 at room temperature)

20 mM [Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂

10 mM nicotinamide adenine dinucleotide

5 mg/ml diaphorase

80 mg/ml sarcosine dehydrognease

12 mg/ml creatinase

24 mg/mL creatininase.

Creatinine Solution

A stock solution of 10 mM creatinine in buffer was, made and dilutedwith buffer to give samples with varying concentration of creatinine.Samples were kept on ice until use.

Wet Testing Protocol

12 μl enzyme mix was placed in an eppendorf, which was then placed on aheat block at 37° C. for 3 minutes. The mix was then added to 1.2 μlcreatinine sample which had also been incubated on the heat block at 37°C. 12 μl of the enzyme/creatinine mix was then immediately placed on theelectrode, and the chronoamperometry test was initiated using amultiplexer (MX452, Sternhagen design) attached to an Autolab (PGSTAT12).

The oxidation current was measured at 0.15 V at 15 time points (0, 14,28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182 and 196 seconds),with a reduction current measured at −0.45 V at the final time point(210 seconds). The transient current was measured for 1 second. Eachsample was tested in duplicate.

Analysis

The output from the GPES software was analysed using the DataAnal2_(—)17 programme. These data were then transferred to the data analysistemplate.

Results

The sensor responses were plotted vs. creatinine concentration. Theslope and intercept for the calibration plot to creatinine at the finaltime point of 196 seconds are given in FIG. 69.

EXAMPLE 19

The aim of the experiment was to demonstrate measurement of glucose inwhole blood using freeze dried sensors prepared with[Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂.

Enzyme Mixture

Enzyme mix was prepared with the following composition:

0.1M Tris (balanced with HCl, pH 9.0 at room temperature) 10% w/v KCl

40 mM [Ru^(III)(Me₃TACN)(acac)(1-MeIm)](NO₃)₂

10 mM nicotinamide adenine dinucleotide

4.2 mg/ml PdR

10 mg/ml glucose dehydrogenase.

Production Dispense and Freeze Drying

0.4 μL/well of the enzyme mixture was dispensed into well 2 of thesensors using the production dispenser. Three other solutions weredispensed into the other wells. These solutions used alternativemediators and the responses of these sensors are not reported here. Thedispensed sensor sheets were then placed into a LS40 freeze drier(Severn Science) for freeze drying. The programme used was Night2. Thesensors formed production batch DEV 345.

Whole Blood Samples

A fresh whole blood sample (Li heparin anticoagulant) was used asreceived. An aliquot of this sample was centrifuged for 5 minutes at2900 RCF (Labnet 1618), and the plasma tested for glucose concentrationusing a Space clinical analyser (Schiappanelli Biosystems Inc). Theglucose concentration was determined to be 5.1 mM.

In addition, aliquots of the initial whole blood sample were spiked with1M glucose solution to obtain higher glucose concentrations. A portionof each spiked aliquot was centrifuged and the plasma tested for glucoseconcentration. Whole blood samples with lower glucose concentration wereobtained by centrifugation of an aliquot, replacement of some plasmawith delipidated serum (Scipac, S 139) and inversion to reconstitute thesample.

Testing Protocol

20 μl of a whole blood sample was used per electrode. On the addition ofsample the chronoamperometry test was initiated using a Uniscanmulti-potentiostat. The oxidation current was measured at 0.15 V at 15time points (0, 14, 28, 42, 56, 70, 84, 98, 112, 126, 140, 154, 168, 182and 196 seconds), with a reduction current measured at −0.45 V at thefinal time point (210). The transient current was measured for 1 second.Each sample was tested in duplicate.

Analysis

The output from the GPES software was analysed using the DataAnal2_(—)17 programme. These data were then transferred to the data analysistemplate.

Results

The sensor responses were plotted vs. glucose concentration (see FIG.70). A good correlation between current and whole blood glucoseconcentration was obtained at 98 seconds (gradient=65.71 nA/sec,intercept=274.18 nA).

EXAMPLE 20

Glucose

In this Example, the whole blood of Example 19 was replaced by saliva.The results are shown in FIG. 71.

1-11. (canceled)
 12. A detection method for measuring an analytecomprising: (a) contacting a sample which contains the analyte with asolution containing an enzyme and a redox mediator of Formula I[M(A)_(w)(B)_(x)(C)_(y)]^(m)(X^(z))_(n)   Formula I wherein M isruthenium or osmium and has an oxidation state of 0, 1, 2, 3 or 4; eachof w, x, and y is an integer independently selected from the integers 1to 4; m is an integer selected from the integers −5 to +4; n is aninteger selected from selected from the integers 1 to 5; z is an integerselected from the integers −2 to +1; A is a monodentate 5- or 6-memberedaromatic ligand containing 1, 2 or 3 nitrogen atoms which is optionallysubstituted by 1 to 8 substituents each selected from the groupconsisting of substituted or unsubstituted alkyl, alkenyl, or arylgroups, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, aryl,alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy,—NH₂, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido,hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino and alkylthio or Ais NCS; B is a bi-, tri-, tetra-, penta- or hexadentate ligand which islinear having the formula R¹RN(C₂H₄NR)_(w)R¹ or cyclic having theformula (RNC₂H₄)_(v), (RNC₂H₄)_(p)(RNC₃H₆)_(q) or[(RNC₂H₄)(RNC₃H₆)]_(s), wherein w is an integer selected from theintegers 1-5, v is an integer selected from the integers 3-6, each of pand q is an integer independently selected from the integers 1-3 wherebythe sum of p and q is 4, 5 or 6, s is either 2 or 3; and each of R andR¹ is independently hydrogen or alkyl; C is a ligand other than B; and Xis a counter ion, wherein the number of coordinating atoms is 6, withthe exception of [Ru^(III)(Me₃tacn)(acac)(py)](NO₃)₂). (b) incubatingthe contacted sample under conditions that cause the enzyme to act onthe analyte; (c) subjecting the incubated sample of step (b) toconditions which result in a change in a measurable signal; and (d)measuring the measurable signal.
 13. A complex according to Formula I[M(A)_(w)(B)_(x)(C)_(y)]^(m)(X^(Z))_(n)   Formula 1 (wherein M isruthenium or osmium and has an oxidation state of 0, 1, 2, 3 or 4; eachof w, x, and y is an integer independently selected from the integers 1to 4; m is an integer selected from the integers −5 to +4; n is aninteger selected from selected from the integers 1 to 5; z is an integerselected from the integers −2 to +1; A is a monodentate 5- or 6-memberedaromatic ligand containing 1, 2 or 3 nitrogen atoms which is optionallysubstituted by 1 to 8 substituents each selected from the groupconsisting of substituted or unsubstituted alkyl, alkenyl, or arylgroups, —F, —Cl, —Br, —I, —NO₂, —CN, —CO₂H, —SO₃H, —NHNH₂, —SH, aryl,alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, —OH, alkoxy,—NH2, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido,hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino and alkylthio; Bis a bi-, tri-, tetra-, penta- or hexadentate ligand which is linearhaving the formula R¹RN(C₂H₄NR)_(w)R¹ or cyclic having the formula(RNC₂H₄)_(v), (RNC₂H₄)_(p)(RNC₃H₆)_(q) or [(RNC₂H₄)(RNC₃H₆)]_(s),wherein w is an integer selected from the integers 1-5, v is an integerselected from the integers 3-6, each of p and q is an integerindependently selected from the integers 1-3 whereby the sum of p and qis 4, 5 or 6, s is either 2 or 3 and each of R and R¹ is independentlyhydrogen or alkyl; C is a ligand other than A or B; and X is a counterion, wherein the number of coordinating atoms is 6, with the exceptionof [Ru^(III)(Me₃tacn)(acac)(Py)](NO₃)₂.
 14. A complex according to claim13 wherein A is selected from the group consisting of NCS, imidazole,pyrazole, thiazole, oxazole, isoquinoline, substituted pyridyl andisomers thereof.
 15. A complex according to claim 13 wherein B is is alinear ligand of formula R¹RN(C₂H₄NR)_(w)R¹, a cyclic ligand of formula(RNC₂H₄)_(v), a cyclic ligand of formula (RNC₂H₄)_(p)(RNC₁H₆)_(q), or acyclic ligand of formula [(RNC₂H₄))(RNC₃H₆)]_(s), wherein w is aninteger selected from 1-3, v is 3 or 4, p and q are integersindependently selected from 1-3, wherein the sum of p and q is 4, and sis 2 or
 3. 16. A complex according to claim 13 wherein C is selectedfrom the group consisting of an amine ligand, CO, CN, a halogen,acetylacetonate (acac), 3-bromoacetylacetonate (Bracac), oxalate,pyridine, and 5-chloro-8-hydroxyquinoline.
 17. A complex according toclaim 13 selected from the group consisting of[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-methylpyridine)]Cl₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3-chloropyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(isonicotinamide)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)pyrazine](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-methoxypyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-dimethylaminopyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-t-butyl-pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(isoquinoline)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(tropolone)(pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(tropolone)(4-t-butyl-pyridine)](NO₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3,4-dimethylpyridine)](CF₃SO₃)₂,and[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3-hydroxypyridine)](NO₃)₂.18. A complex according to claim 13 selected from the group consistingof[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](PF6)2andRu^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](NO₃)₂.19. The detection method according to claim 12 wherein ligand A isselected from the group consisting of NCS, imidazole, pyrazole,thiazole, oxazole, isoquinoline, substituted pyridyl, and isomersthereof.
 20. The detection method according to claim 12 wherein ligand Ais guanine, adenine, a 5-membered heteroaromatic comprising threenitrogen atoms in the ring, a 6-membered heteroaromatic comprising threenitrogen atoms in the ring, imidazole, pyrazole, thiazole, oxazole, orisomers thereof.
 21. The detection method according to claim 12 whereinligand A is substituted by one or more substituents selected from thegroup consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆alkynyl and halogen.
 22. The detection method according to claim 21wherein ligand A is substituted by one or more substituents selectedfrom the group consisting of methyl, ethyl, propyl, iso-propyl, butyl,t-butyl, methoxy, ethoxy, ethenyl, propenyl, butenyl, ethynyl andpropynyl.
 23. The detection method according to claim 12 wherein ligandB is a linear ligand of formula R¹RN(C₂H₄NR)_(w)R¹, a cyclic ligand offormula (RNC₂H₄)_(v), a cyclic ligand of formula(RNC₂H₄)_(p)(RNC₁H₆)_(q), or a cyclic ligand of formula[(RNC₂H₄))(RNC₃H₆)]_(s), wherein w is an integer selected from 1-3, v is3 or 4, p and q are integers independently selected from 1-3, whereinthe sum of p and q is 4, and s is 2 or
 3. 24. The detection methodaccording to claim 12 wherein B is1,4,7-trimethyl-1,4,7-triazacyclononane,1,1,4,7,10,10-hexamethyltriethylenetetramine,1,2-dimethylethylenediamine or 1,1,2,2-tetramethylethylenediamine. 25.The detection method according to claim 12 wherein ligand C is selectedfrom the group consisting of NH₂, CO, CN, a halogen, acetylacetonate(acac), 3-bromo-acetylacetonate (Bracac), oxalate, pyridine or5-chloro-8-hydroxyquinoline.
 26. The detection method according to claim12 wherein the complex of Formula I is[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-methylpyridine)]Cl₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3-chloropyridine)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(isonicotinamide)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)pyrazine](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-methoxypyridine)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(4-dimethylaminopyridine)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(44-butyl-pyridine)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(isoquinoline)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(tropolone)(pyridine)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(tropolone)(4-t-butyl-pyridine)](N0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3,4-dimethylpyridine)](CF₃S0₃)₂,[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(3-hydroxypyridine)](N0₃),or[Ru^(III)(1,4,8,11-tetramethyl-1,4,8,11-tetra-azacyclotetradecane)(NCS)₂](ClO₄).27. The detection method according to claim 12 wherein the complex ofFormula I is[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](PF₆)₂or[Ru^(III)(1,4,7-trimethyl-1,4,7-triazacyclononane)(acac)(N-methylimidazole)](NO₃)₂.28. The complex according to claim 13 wherein ligand A is guanine,adenine, 5-membered heteroaromatic comprising three nitrogen atoms inthe ring, 6-membered heteroaromatic comprising three nitrogen atoms inthe ring, imidazole, pyrazole, thiazole, oxazole, or isomers thereof.29. The complex according to claim 13 wherein ligand A is substituted byone or more substituents selected from the group consisting of C₁-C₆alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl and halogen.
 30. Thecomplex according to claim 13 wherein ligand A is substituted by one ormore substituents selected from the group consisting of methyl, ethyl,propyl, iso-propyl, butyl, t-butyl, methoxy, ethoxy, ethenyl, propenyl,butenyl, ethynyl and propynyl.
 31. The complex according to claim 13wherein B is 1,4,7-trimethyl-1,4,7-triazacyclononane,1,1,4,7,10,10-hexamethyltriethylenetetramine,1,2-dimethylethylenediamine or 1,1,2,2-tetramethylethylenediamine.